Histone Deacetylase (HDAC)
DNA in eukaryotic cells is tightly complexed with proteins (histones) to form chromatin. Histones are small, positively charged proteins which are rich in basic amino acids (positively charged at physiological pH), which contact the phosphate groups (negatively charged at physiological pH) of DNA. There are five main classes of histones, H1, H2A, H2B, H3, and H4. The amino acid sequences of histones H2A, H2B, H3, and H4 show remarkable conservation between species, whereas H1 varies somewhat, and in some cases is replaced by another histone, e.g., H5. Four pairs of each of H2A, H2B, H3, and H4 together form a disk-shaped octomeric protein core, around which DNA (about 140 base pairs) is wound to form a nucleosome. Individual nucleosomes are connected by short stretches of linker DNA associated with another histone molecule (e.g., H1, or in certain cases, H5) to form a structure resembling a beaded string, which is itself arranged in a helical stack, known as a solenoid.
The majority of histones are synthesised during the S phase of the cell cycle, and newly synthesised histones quickly enter the nucleus to become associated with DNA. Within minutes of its synthesis, new DNA becomes associated with histones in nucleosomal structures.
A small fraction of histones, more specifically, the amino side chains thereof, are enzymatically modified by post-translational addition of methyl, acetyl, or phosphate groups, neutralising the positive charge of the side chain, or converting it to a negative charge. For example, lysine and arginine groups may be methylated, lysine groups may be acetylated, and serine groups may be phosphorylated. For lysine, the —(CH2)4—NH2 sidechain may be acetylated, for example by an acetyltransferase enzyme, to give the amide —(CH2)4—NHC(═O)CH3. Methylation, acetylation, and phosphorylation of amino termini of histones which extend from the nucleosomal core affect chromatin structure and gene expression. (See, for example, Spencer, V. A. and Davie, J. R., 1999, Gene, Vol. 240(1), pp. 1-12).
Acetylation and deacetylation of histones is associated with transcriptional events leading to cell proliferation and/or differentiation. Regulation of the function of transcription factors is also mediated through acetylation. Recent reviews of histone deacetylation include: Kouzarides, T., 1999, “Histone acetylases and deacetylases in cell proliferation,” Curr. Opin. Genet. Dev., Vol. 9, No. 1, pp. 40-48; Pazin, M. J., et al., 1997, “What's up and down with histone deacetylation and transcription?,” Cell, Vol. 89, No. 3, pp. 325-328.
The correlation between the acetylation status of histones and the transcription of genes has been known for over 30 years (see, for example, Howe, L., et al., 1999, Crit. Rev. Eukaryot. Gene Expr., Vol. 9(3-4), pp. 231-243). Certain enzymes, specifically acetylases (e.g., histone acetyltransferase, HAT) and deacetylases (e.g., histone deacetylase, HDAC), which regulate the acetylation state of histones have been identified in many organisms and have been implicated in the regulation of numerous genes, confirming the link between acetylation and transcription. See, for example, Davie, J. R., 1998, “Covalent modifications of histones: expression from chromatic templates,” Curr. Opin. Genet. Dev., Vol. 8, pp. 173-178. In general, histone acetylation correlates with transcriptional activation, whereas histone deacetylation is associated with gene repression.
A growing number of histone deacetylases (HDACs) have been identified, including HDAC1 through HDAC11 (see, for example, Ng, H. H. and Bird, A., 2000, Trends Biochem. Sci., Vol. 25(3), pp. 121-126). A number of yeast histone deacetylases and plant histone deacetylases have also been identified. The first deacetylase, HDAC1, was identified in 1996 (see, for example, Taunton, J., et al., 1996, Science, Vol. 272, pp. 408 411). Subsequently, two other nuclear mammalian deacetylases were found, HDAC2 and HDAC3. See, for example: Yang, W. M., et al., 1996, Proc. Natl. Acad. Sci. USA, Vol. 93, pp. 12845-12850; Yang, W. M., et al., 1997, J. Biol. Chem., Vol. 272, pp. 28001-28007; Emiliani, S., et al., 1998, Proc. Natl. Acad. Sci. USA, Vol. 95, p. 2795-2800; Grozinger et al., 1999, Proc. Natl. Acad. Sci. USA, Vol. 96, pp. 4868-4873; Kao et al., 2000, Genes & Dev., Vol. 14, p. 55-66; Van den Wyngaert et al., 2000, FEBS, Vol. 478, pp. 77-83.
HDACs function as part of large multi-protein complexes, which are tethered to the promoter and repress transcription. Well characterised transcriptional repressors such as Mad (Laherty, C. D., et al., 1997, Cell, Vol. 89(3), pp. 349-356), pRb (Brehm, A., et al., 1998, Nature, 1998, Vol. 391, pp. 597-601), nuclear receptors (Wong, J., et al., 1998, EMBO J., Vol. 17(2), pp. 520-534) and YY1 (Yang, W. M., et al., 1997, J. Biol. Chem., Vol. 272, pp. 28001-28007) associate with HDAC complexes to exert their repressor function.
The Role of HDAC in Cell Proliferation
The study of inhibitors of histone deacetylases indicates that these enzymes play an important role in cell proliferation and differentiation. The inhibitor Trichostatin A (TSA) (Yoshida, M., et al., 1990, J. Biol. Chem., Vol. 265(28), pp. 17174-17179) causes cell cycle arrest at both G1 and G2 phases (Yoshida, M., Beppu, T., 1988, Exp. Cell. Res., Vol. 177, pp. 122-131), reverts the transformed phenotype of different cell lines, and induces differentiation of Friend leukaemia cells and others (Yoshida, M., et al., 1990, J. Antibiot. (Tokyo), Vol. 43(9), pp. 1101-1106). TSA (and SAHA) have been reported to inhibit cell growth, induce terminal differentiation, and prevent the formation of tumours in mice (Finnin et al., 1999, Nature, Vol. 401, pp. 188-193). Cell cycle arrest by TSA correlates with an increased expression of gelsolin (Hoshikawa, Y., et al., 1994, Exp. Cell. Res., Vol. 214(1), pp. 189-197), an actin regulatory protein that is down regulated in malignant breast cancer (Mielnicki, L. M., et al., 1999, Exp. Cell. Res., Vol. 249(1), pp. 161-176). Similar effects on cell cycle and differentiation have been observed with a number of deacetylase inhibitors (Kim et al., 1999, Oncogene, Vol. 18(15), pp. 2461-2470).
The clear involvement of HDACs in the control of cell proliferation and differentiation suggests that aberrant HDAC activity may play a role in cancer. The most direct demonstration that deacetylases contribute to cancer development comes from the analysis of different acute promyelocytic leukemias (APL). In most APL subjects, a translocation of chromosomes 15 and 17 (t(15;17)) results in the expression of a fusion protein containing the N-terminal portion of PML gene product linked to most of RARα (retinoic acid receptor). In some cases, a different translocation (t(11;17)) causes the fusion between the zinc finger protein PLZF and RARα. In the absence of ligand, the wild type RARα represses target genes by tethering HDAC repressor complexes to the promoter DNA. During normal hematopoiesis, retinoic acid (RA) binds RARα and displaces the repressor complex, allowing expression of genes implicated in myeloid differentiation. The RARα fusion proteins occurring in APL subjects are no longer responsive to physiological levels of RA and they interfere with the expression of the RA-inducible genes that promote myeloid differentiation. This results in a clonal expansion of promyelocytic cells and development of leukaemia. In vitro experiments have shown that TSA is capable of restoring RA-responsiveness to the fusion RARα proteins and of allowing myeloid differentiation. These results establish a link between HDACs and oncogenesis and suggest that HDACs are potential targets for pharmaceutical intervention in APL subjects. (See, for example, Kitamura, K., et al., 2000, Br. J. Haematol., Vol. 108(4), pp. 696-702; David, G., et al., 1998, Oncogene, Vol. 16(19), pp. 2549-2556; Lin, R. J., et al., 1998, Nature, Vol. 391(6669), pp. 811-814).
Furthermore, different lines of evidence suggest that HDACs may be important therapeutic targets in other types of cancer. Cell lines derived from many different cancers (prostate, colorectal, breast, neuronal, hepatic) are induced to differentiate by HDAC inhibitors (Yoshida, M. and Horinouchi, S., 1999, Ann. N. Y. Acad. Sci., Vol. 886, pp. 23-36). A number of HDAC inhibitors have been studied in animal models of cancer. They reduce tumour growth and prolong the lifespan of mice bearing different types of transplanted tumours, including melanoma, leukaemia, colon, lung and gastric carcinomas, etc. (Ueda, H., et al., 1994, J. Antibiot. (Tokyo), Vol. 47(3), pp. 315-323; Kim et al., 1999, Oncogene, Vol. 18(15), pp. 2461-2470).
Psoriasis is a common chronic disfiguring skin disease which is characterised by well-demarcated, red, hardened scaly plaques: these may be limited or widespread. The prevalence rate of psoriasis is approximately 2%, i.e., 12.5 million sufferers in the triad countries (US/Europe/Japan). While the disease is rarely fatal, it clearly has serious detrimental effects upon the quality of life of the subject: this is further compounded by the lack of effective therapies. Present treatments are either ineffective, cosmetically unacceptable, or possess undesired side effects. There is therefore a large unmet clinical need for effective and safe HDACis for this condition.
Psoriasis is a disease of complex etiology. Whilst there is clearly a genetic component, with a number of gene loci being involved, there are also undefined environmental triggers. Whatever the ultimate cause of psoriasis, at the cellular level, it is characterised by local T-cell mediated inflammation, by keratinocyte hyperproliferation, and by localised angiogenesis. These are all processes in which histone deacetylases have been implicated (see, e.g., Saunders, N. et al, 1999, Cancer Res., Vol. 59, No. 2 pp. 399-404; Bernhard, D. et al., 1999, FASEB J., Vol. 13, No. 14, pp. 1991-2001; Takahashi et al., 1996, J. Antibiot. (Tokyo), Vol. 49, No. 5, pp. 453-457; Kim et al., 2001, Nature Medicine, Vol. 7, No. 4, pp. 437-443). Therefore HDAC inhibitors may be of use in therapy for psoriasis. Candidate HDACis may be screened, for example, using proliferation assays with T-cells and/or keratinocytes.
HDAC Inhibitors
One important class of HDAC inhibitors are carbamic acid compounds comprising a sulfonamide linkage, as described, for example, in Watkins, C., et al., 2002, published international (PCT) patent application number WO 02/30879. An especially promising compound is N-hydroxy-3-(3-phenylsulfamoyl-phenyl)-acrylamide (referred to herein as PXD-101).

Many potentially useful HDACis suffer from one or more formulation problems, for example, low solubility in aqueous solutions, the need to employ unsuitably high or low pH in order to effect HDACi solubilisation, physical and/or chemical instability in aqueous solutions, physical and/or chemical instability upon later dilution, etc. Compounds such as PXD-101 also suffer from these and other problems.
Thus, one aim of the present invention is the provision of improved pharmaceutical compositions (e.g., formulations and pre-formulations) which comprise PXD-101 or structurally similar compounds, which address one or more of the above and other problems.
The inventors have found particular combinations of ingredients which, surprisingly and unexpectedly, yield pharmaceutical compositions that have greatly improved properties.
These pharmaceutical compositions offer one or more of the following advantages:
(a) a greater concentration of HDACi;
(b) increased stability when in a concentrated liquid form (e.g., for storage);
(c) increased stability when in a diluted liquid form (e.g., when ready for administration);
(d) the ability to provide the composition as, for example, a ready-to-use solution, a concentrate for extemporaneous dilution, and/or a lyophilate/lyophilisate.
A number of patents and publications are cited herein in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided herein. Each of these references is incorporated herein by reference in its entirety into the present disclosure.